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EP2948467A1 - Procédé pour la synthèse à base de phase solide de conjugués de nucléoside à pont phosphate - Google Patents

Procédé pour la synthèse à base de phase solide de conjugués de nucléoside à pont phosphate

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Publication number
EP2948467A1
EP2948467A1 EP13701961.8A EP13701961A EP2948467A1 EP 2948467 A1 EP2948467 A1 EP 2948467A1 EP 13701961 A EP13701961 A EP 13701961A EP 2948467 A1 EP2948467 A1 EP 2948467A1
Authority
EP
European Patent Office
Prior art keywords
nucleoside
analog
compound
methyl
phosphate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP13701961.8A
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German (de)
English (en)
Inventor
Ivo SARAC
Chris Meier
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Universitaet Hamburg
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Universitaet Hamburg
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • the invention relates to a method for the solid-phase based synthesis of phosphate-bridged nucleoside conjugates, in particular of oligonucleosides or oligonucleotides.
  • Phosphate-bridged nucleoside conjugates are of great importance in nature. They are not only significantly involved in metabolic-energetic processes, but are present in nearly all bio syntheses as metabolites.
  • Examples for such phosphate-bridged nucleoside conjugates are nucleoside di- and -triphosphates, e.g. the naturally occurring ribo- and deoxyribonucleoside triphosphates (NTP's and dNTP's), oligonucleosides and oligonucleotides, dinucleoside-polyphosphates, NDP sugars or sugar nucleotides, and nucleoside conjugates with peptides etc.
  • NTP's and dNTP's Naturally occurring ribo- and deoxyribonucleoside triphosphates (NTP's and dNTP's), for ex- ample, represent basic building blocks for the enzymatically catalyzed RNA and DNA synthesis in vivo and in vitro, while their analogs have an enormous potential as inhibitors in many biological processes (e.g. processes in which DNA polymerases are involved) or as chemo- therapeutics. For this reason, there is great interest in a synthetic access to these compounds. However, not only the synthesis of nucleoside triphosphates, but also their isolation is a big problem. Further, nucleoside triphosphates are susceptible for hydrolysis due to their energy- rich anhydride bonds.
  • Oligonucleotides are also of great practical interest, because these compounds have a wide range of applications in e.g. genetic testing, research, and forensics.
  • the comparatively small nucleic acids can be manufactured with a user-specified sequence, and so are very important for the synthesis of artificial gene, the polymerase chain reaction (PCR), for DNA sequencing, library construction and as molecular probes.
  • PCR polymerase chain reaction
  • Oligonucleotides are often synthesized in 3 -5' direction on a solid-phase using phosphoramidite building blocks derived from protected 2'- deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. locked nucleic acids (LNA) (see e.g. McBride, LJ, Caruthers, MH, 1983, Tetrahedron Letters, 24, 245 248; Beaucage, S. L., Iyer, R. P., 1992, Tetrahedron 48, 2223- 2311).
  • LNA locked nucleic acids
  • the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product.
  • the process has been fully automated since the late 1970s and uses the co called solid-phase synthesis ap- proach.
  • the product Upon the completion of the chain assembly, the product is released from the solid- phase to solution, deprotected, and collected.
  • the occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized.
  • Products are often isolated by high-performance liquid chromatography (HPLC) to obtain the desired oligonucleotides in high purity.
  • synthetic oligonucleotides are single- stranded DNA or RNA molecules around 15-25 bases in length.
  • WO 2010/015245 Al and WO 2010/127666 Al both disclose methods for the synthesis of phosphate-bridged nucleoside conjugates using so-called cj ⁇ /oSaligenyl(cyc/oSal) nucleoside phosphate triesters.
  • a cycloSal nucleoside is bound to a linker and immobilized subsequently via the linker on a solid phase or bound to a linker already bound to the solid-phase. Subsequently a nucleophile is reacted with the immobilized cycloSal nucleoside.
  • Object of the present invention is to improve the current methods for preparing phosphate- bridged nucleotide bioconjugates, in particular the preparation of oligonucleosides and oligonucleotides.
  • the object is solved by the method of claim 1. Preferred embodiments are specified in the dependent claims.
  • R 1 is a nucleoside, nucleotide, polynucleoside, polynucleotide or an analog thereof
  • R 2 is an organic compound or phosphate or pyrophosphate, or a residue thereof
  • X being H, an electron acceptor or an electron acceptor precursor and Y being halogen, preferably CI or Br, or -NR 3 R 4 , wherein R 3 and R 4 are, independently, substituted or unsubstituted alkyl or substituted or unsubstituted aryl, preferably substituted or unsubstituted Ci-Cio alkyl or substituted or unsubstituted C6-C20 aryl, and wherein the compound II may be substituted one or more times with X,
  • R 1 and X being as defined above, Z being O or S, SP being the solid phase and (L) being the optional linker, and
  • phosphate-bridged nucleoside conjugates is meant herein a compound of the general formula O
  • R 1 is a nucleoside, nucleotide, polynucleoside, polynucleotide or an analog thereof.
  • the nucleoside, nucleotide, polynucleoside, polynucleotide or an analog thereof is preferably bound to the phosphate atom via an oxygen atom of the sugar component or sugar component analog, in case of a polynucleoside or polynucleotide preferably via an oxygen atom of a terminal sugar component or sugar component analog, e.g. via an oxygen atom at the 2', 3' or 5' C-atom, preferably the 5' C-atom, of the sugar component, e.g. ribose.
  • R 2 is any or- ganic compound, preferably a phosphorylated organic compound, or phosphate or pyrophosphate, or a residue thereof.
  • R 2 is a compound or compound residue, or a component analogous to said compound or compound residue, which is present in a living cell, for example an alcohol, a sugar, a steroid, a lipid, a nucleoside, a nucleoside mono-, di- or triphosphate, phosphate or pyrophosphate, or a residue thereof.
  • bioconjugates is used for such preferred phosphate-bridged nu- cleoside conjugates.
  • cyc/oSal nucleotide or "cyc/o Saligenyl nucleotide” as used herein means compounds according to the following general formula IV
  • R 1 is a nucleoside, nucleotide, polynucleoside, polynucleotide or an analog thereof, and wherein Z is oxygen (O) or sulfur (S).
  • the term covers cyclic phosphate triester derivatives, in which a salicyl alcohol (saligenol) is diesterified in a cyclic manner with a (mono)phosphate residue, e.g. a phosphate residue bound at the 5 '-atom of a ribose or deoxyri- bose of a nucleoside, nucleotide, polynucleoside, polynucleotide or an analog thereof.
  • phrases (L) as used herein is understood to mean an organic compound by which another compound, e.g. a compound according to the above formula IV, is covalently bound to a solid phase.
  • a linker usually has at least two functional groups e.g., carboxyl groups -COOH, and is covalently linked with both the compound and the solid phase, thus serving as connecting piece and/or spacer between the compound and the solid phase.
  • Linker compounds are known in the prior art.
  • An example of a linker is a succinyl linker according to formula (V)
  • linker In a chemical formula a linker is represented by the letter “L”. An optional linker is represented by the letter “L” in parentheses, i.e. "(L)”.
  • organic compound is any compound having bonds of carbon with carbon and with other elements (with the exception of carbon dioxide, carbon monoxide, carbonic acid and its carbonates, and cyanides, isocyanides, cyanates and isocyanates of metals).
  • organic compounds are carbohydrates, i.e.
  • organic compound may, for example, be a phosphorylated organic compound.
  • a “heterocycle” is a cyclic compound with ring-forming atoms of at least two different chemical elements.
  • the term means a ring-forming organic component in the ring structure of which at least one carbon atom is replaced by another element, i.e. a heteroatom, for example nitrogen, oxygen, phosphor and/or sulfur.
  • a ring structure can consist of one or more rings connected with each other and may contain one or more identical or different hetero- atoms.
  • the term "nucleophile” as used herein has the usual meaning known by the skilled person.
  • a nucleophile means a molecule containing a negatively polarized region, a negatively polarized functional group or a free electron pair, generally in an energy rich orbital.
  • the term also covers molecules being nucleophile, i.e. relatively electron richer in relation to a reaction partner or to a region of the reaction partner.
  • the reaction partner also is termed electrophile, because it assumes electrons from the nucleophile.
  • Nucleophiles may form covalent bonds by providing electrons to a reaction partner. The electrons necessary for the bond are generally from the nucleophile alone.
  • Nucleophiles can be, and are preferably, negatively charged (anions).
  • nucleophile reagents examples are carbanions, anions, Lewis bases, aromatics, alcohols, amines, e.g. amino acids, and compounds with olefmic dou- ble bonds.
  • the strength of the nucleophilicity depends, for example, on the reaction partner, the basicity, the solvent and sterical factors. The factors affecting the nucleophilicity of a compound are well known to the skilled person, and he can easily determine their nucleophilic properties.
  • the nucleophilicity of a molecule will advantageously be related to the most nucleophilic atom or the most nucleophilic functional group.
  • a cycloSal nucleotide ac- cording to the above general formula (IV) is employed as an electrophile
  • the electrophilicity of the phosphorus atom can be controlled via the substituent X at the cycloSal aromatic ring (s. C. Meier, J. Renze, C. Ducho, J. Balzarini, Curr. Topics in Med. Chem. 2002, 2, 1111-1121, the disclosure of which is incorporated herein by reference in its entirety).
  • the electrophilicity can be reduced, acceptor substitu- ents, however, increase the reaction rate of the initial reaction, i.e. the cycloSal ring opening.
  • an "electron acceptor” is a compound, a region of a compound or a functional group, drawing electrons to it and thereby causing a charge displacement, i.e. a polarization, in a compound.
  • Preferred esters as electron acceptors are esters whose ester group is situated as close as possible to, preferably directly at, the aromatic ring.
  • Ketones preferred as electron acceptors are ketones whose keto group is situated as close as possible to, preferably directly at, the aromatic ring.
  • An "electron acceptor precursor” is a compound which can be activated, i.e. converted into an electron acceptor, by cleaving off a masking group.
  • Esters are compounds containing the ester group R'-COO-R", wherein R' and R" may be any substituted or unsubstituted, branched- or linear hydrocarbon residues, for example alkyl residues or aryl residues.
  • Ketones are compounds containing the keto group R'-CO-R", wherein R' and R" are any sub- stituted or unsubstituted, branched or linear hydrocarbon residues, for example alkyl residues or aryl residues.
  • nucleoside is meant herein organic molecules consisting of a sugar residue (sugar component) and an organic base (base component), e.g. a heterocyclic organic base, in particular a nitrogen containing heterocyclic organic base, being connected via a glycosidic bond.
  • the sugar residue often is a pentose, e.g. deoxyribose or ribose, but may also be another sugar, e.g. a C 3 , C 4 or C 6 sugar.
  • nucleoside is meant a compound according to the general formula (VI)
  • B is a nitrogen containing heterocyclic base, e.g. a nucleobase
  • R 8 and R 9 are, independent from each other, H or OH.
  • the term also encompasses LNA (locked nucleic acid) nucleosides, i.e. nucleosides, wherein the ribose moiety contains a bridge connecting the 2' oxygen and 4' carbon, thereby "locking" the ribose in the 3'-endo (North) conformation.
  • LNA locked nucleic acid
  • nucleobase is meant an organic base occurring in RNA and/or DNA.
  • Naturally occurring nucleobases are purines (R) and pyrimidines (Y).
  • purines are guanine (G) and adenine (A)
  • examples for pyrimidines are cytosine (C), thymine (T) and uracil (U).
  • Phos- phorylated nucleoside for example nucleoside monophosphate (NMP), nucleoside diphosphate (NDP) and nucleoside triphosphate (NTP) are also termed nucleotides.
  • nucleoside analog is meant herein a compound, which naturally does not occur in a living cell of e.g. a human body, but is structurally similar to a nucleoside naturally occurring in a living cell of e.g.
  • a sugar analog can, for example, be a carbocycle wherein the ring oxygen atom is replaced by a CH 2 group.
  • base analogs are 7-deazapurines, isoadenine, hypoxanthine, halogenated pyrimidines (like 5-fluoruracil) etc.
  • a nucleoside analog can itself be a nucleoside. It can, however, also be another compound with the above properties, for example a compound of a heterocyclic base and an acyclic residue and/or a residue that is not a sugar, or a compound of a carbocyclic compound and a sugar residue, or a compound composed of a carbocycle replacing the sugar component, e.g.
  • nucleoside analogs are either itself nucleosides in the above sense or structurally and/or functionally analogous to nucleosides. Since the nucleoside analogs may not necessarily contain a sugar or base component in a narrower sense, it is also spoken of a component analogous to the base component (base analog) or a component analogous to a sugar component (sugar analog).
  • nucleoside analogs are, for example, AZT (3'-azido-2',3'-dideoxythimi- dine, azidothymidine), 2',3'-dideoxyinosine (didanosine), 2',3'-dideoxycytidine (zalticabine) and 2-amino-9-((2-hydroxyethoxy)methyl)-lH-purine-6(9H)-one (acyclovir).
  • Nucleoside phosphonates can also be nucleoside analogs.
  • polynucleoside refers to polymers composed of a sequence of two or more nucleoside units linked by internucleoside bonding groups ("backbone” linkages).
  • backbone linkages i.e. polynucleotides
  • nucleoside polymers linked by structures other than phosphodi- ester bonds Such bonds may be modified phosphodiester linkages, e.g.
  • phosphodiester linkages in which one of the non-bridging phosphate oxygens in the linkage is replaced with sulfur, methyl or other atoms or groups, or non-phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl- (e.g. methyl-) and arylphosphonate, phosphoramidate, phosphotri- ester, alkyl- (e.g. methyl-) and arylphosphonothioate, aminoalkylphosphonate, aminoalkyl- phosphonothioate, phosphorofluoridate, boranophosphate, silyl, formacetal, thioformacetal, morpholino and peptide-based linkages.
  • Chimeric compounds having a mixture of such linkages and/or compounds consisting of or comprising LNA nucleosides are also encompassed by the term "polynucleoside".
  • polynucleoside analog refers to a molecule comprising at least one nucleoside analog
  • oligonucleotide analog refers to a molecule compris- ing at least one nucleotide analog.
  • DNA or "deoxyribonucleic acid” denotes polynucleotides, wherein the sugar component is deoxyribose.
  • the term in particular comprises polynucleotides wherein the sugar component is deoxyribose, the internucleoside linkages are phosphodiester linkages, and the base components are selected from the group consisting of adenine, cytosine, guanine and thymine.
  • R A or "ribonucleic acid” means denotes polynucleotides, wherein the sugar component is ribose.
  • the term in particular comprises polynucleotides wherein the sugar com- ponent is ribose, the internucleoside linkages are phosphodiester linkages, and the base components are selected from the group consisting of adenine, cytosine, guanine and uracil.
  • oligonucleoside refers to relatively short polynucleosides. In particular the term refers to molecules consisting of not more than 250 nucleoside units, preferably 2-200, 2-150, or 2-100 nucleosides.
  • oligonucleotide refers to oligonucleosides wherein the nucleosides are linked by phosphodiester backbone linkages.
  • oligonucleoside analog refers to a molecule comprising at least one nucleoside analog, the term “oligonucleotide analog” to a molecule comprising at least one nucleotide analog.
  • glycosyl phosphate is meant a phosphorylated glycosyl residue.
  • the glycosyl residue may, for example, be phosphorylated at the C 1 atom, but may alternatively or additionally be phosphorylated at other positions, e.g. a C6 atom.
  • a "glycosyl” is a compound with a functional group derived from a sugar by elimination of hemiacetal hydroxyl group.
  • glycosyl- 1 -phosphates examples include: glucose- 1 -phosphate, mannose-1 -phosphate, galactose- 1- phosphate, 2-N-acetyl-glucosamine-l -phosphate, 6-deoxygulose-l -phosphate, 2-N-acetyl- galactosamine- 1 -phosphate, D-fucose- 1 -phosphate and L-fucose- 1 -phosphate; each in the a or ⁇ configuration at the anomeric center (in case of mannose there is only the a form).
  • An example for glycosyl-6-phosphates is glucose-6-phosphate.
  • alkyl refers to branched or straight-chain (unbranched, linear), satu- rated or unsaturated, aliphatic (non-aromatic) hydrocarbon groups, for example methyl, ethyl, n-propyl, i-propyl, n-butyl, t-butyl, i-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl groups.
  • the term thus encompasses alkenyls and alkynyls.
  • the term also comprises the term "cycloal- kyl", meaning mono-, bi- or polycyclic aliphatic hydrocarbon groups.
  • cycloalkyl includes, for example, cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl- cyclopentyl and cyclohexyl.
  • alkyl also covers the term “heteroalkyl”, being an alkyl wherein at least one carbon atoms is replaced by a "heteroatom”, i.e. a non-carbon atom, e.g. oxygen, sulfur, nitrogen or phosphor.
  • Ci-Cio alkyl means an alkyl having 1 , 2, 3, 4, 5, 6, 7, 8, 9 or IO C atoms.
  • aryl refers to monocyclic, bicyclic and polycyclic substituted or un- substituted aromatic hydrocarbons, including a single ring or multiple aromatic rings fused or linked together where at least one part of the fused or linked rings forms the conjugated aromatic system.
  • the aryl groups can typically have from 6 to 20 or more carbon atoms and can include, but are not limited to, e.g. phenyl, naphthyl, biphenyl, anthranyl, tetrahydronaphthyl, phenanthryl, indene, benzonaphthyl, fluorenyl, and carbazolyl.
  • heteroaryl meaning aryls containing at least one heteroatom within the ring structure.
  • C 6 -C 20 aryl means an aryl having 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 C-atoms, including C-atoms of any substituents.
  • amine is meant compounds of the type R-NH 2 , NH-R 2 , N-R 3 and N-P + , R representing a substituted or unsubstituted alkyl or aryl residue, wherein, in case of multiple residues, these can be different or the same.
  • the residues may be closed to a ring, so that the term also encompasses cyclic amines.
  • An amine used as nucleophile has preferably the structure R-NH 2 or NH-R 2 .
  • protecting group PG as used herein is meant a molecule or molecule residue, which blocks a functional group within a compound during a reaction at another site of the compound and which prevents unwanted (side) reactions.
  • a protecting group can ideally be introduced under the mildest conditions possible, is stable under the subsequent conditions, and can mildly be cleaved off after the reaction.
  • Protecting groups are well known to the skilled person, so that he or she will easily find a suitable protecting group, if necessary, after routine experimentation. Examples for a protecting group are the methyl, acetyl, 2-cyanoethyl, and benzoyl group.
  • An OH group can, for example, be protected by O methylation or O
  • OPG for example, is a protecting group bound to an oxygen atom.
  • Protecting groups are, for example, described in Peter G.M. Wuts, Theodora W. Greene: Green's
  • halogen refers to a group of elements comprising fluorine (F), chlorine (CI), bromine (Br), and iodine (I). As used herein, the term relates in particular to a halogen residue.
  • the Z at the P atom of the cycloSal moiety could again be O.
  • the method of the invention has a broad applicability to a wide variety of compounds. With the method of the invention any phosphate-bridged nucleoside conjugate can efficiently be prepared.
  • nucleoside diphosphate glycopyranoses examples are nucleoside diphosphate glycopyranoses, sugar-nucleoside bioconju- gates, nucleoside di- and nucleoside triphosphates, dinucleoside monophosphates, dinucleoside polyphosphates, or nucleoside analogs, which may be employed as "prodrugs", i.e. precursors of active agents later releasing the active agent.
  • the method of the invention is especially useful for the preparation of poly- or oligonucleosides, in particular RNA, DNA and/or LNA poly- or oligonucleosides, e.g. RNA, DNA and/or LNA poly- or oligonucleotides.
  • R 3 and R 4 are, independently, substituted or unsubstituted alkyl or aryl, preferably Ci Cio alkyl or C6-C20 aryl, e.g. both isopropyl as in formula (Ilbl)
  • Hal are CI and Br.
  • the solid phase may, optionally via a linker, for example be bound to an oxygen atom at the 2'- or 3'-C-atom of the sugar component, e.g. a pentose, or sugar component analog of R 1 .
  • a linker for example be bound to an oxygen atom at the 2'- or 3'-C-atom of the sugar component, e.g. a pentose, or sugar component analog of R 1 .
  • Other possibilities also exist, e.g. oxygen or nitrogen atoms at other sites of the nucleoside or nucleoside analog.
  • OH groups or, as the case may be, other functional groups at which a chemical reaction is to be avoided can be protected with a protecting group.
  • the solid phase or the linker are co- valently bound to an oxygen atom of a sugar component of R 1 , preferably an oxygen atom bound to a 2'- or 3' C atom of the sugar component, or to an oxygen atom of a component analogous to a sugar component of R 1 , and wherein the residue of formula Ila
  • the solid phase or the linker may, for example, be linked to an oxygen atom bound at the 2'- or 3' C atom of a ribose or deoxyribose of the first nucleoside, i.e.
  • compound III is a compound according to formula Ilia
  • Z is, independently for each occurrence, O or S, preferably O
  • B is, independently for each occurrence, a heterocycle, preferably a nitrogen containing heterocycle, especially preferred a nucleobase
  • R 6 is, independently for each occurrence, H, OPG, PG being a protecting group
  • R 7 is, independently for each occurrence, H or OPG, PG being a protecting group
  • n is an integer > 0.
  • B is, independently for each occurrence, one of the nucleobases guanine, adenine, cytosine, thymine or uracil.
  • B can, for example, also be a nucleobase analog.
  • B is, independently for each occurrence, one of the nucleobases guanine, adenine, cytosine, thymine or uracil and Z is O or S, preferably O.
  • R 1 is preferably selected from the group consisting of oligonucleoside, oligonucleotide, oligonucleoside analog, oligonucleotide analog, adenosine, guanosine, cytidine, thymidine, uridine, deoxyadenosine, deoxyguanosine, inosine, deoxycytidine, deoxyuridine,
  • R 1 is an oligonucleoside, oligonucleotide, oligonucle
  • the solid phase or the linker may alternatively be covalently bound to a nitrogen atom of a base component of the nucleoside, nucleotide, oligonucleoside or oligonucleotide, or to a nitrogen atom of a component analogous to a base component of the nucleoside analog, nucleotide analog, oligonucleoside analog or oligonucleotide analog of R 1 .
  • the aromatic ring in compound can be one or more times substituted with X, wherein the substituents can be the same or different.
  • the compound according to formula (II) can also be substituted at the C atom 7 (for the numbering see formula IV), for example with methyl, i- propyl, tert-butyl or other alkyl substituents.
  • the aromatic ring can have further substituents apart from X, for example alkyl or aryl substituents.
  • the nucleophile is selected from the group consisting of phosphate, pyrophosphate, glycosyl phosphate, nucleoside, nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate, nucleoside analog, nucleoside monophosphate analog, nucleoside diphosphate analog, nucleoside triphosphate analog, a- deprotonated glycosyl, deprotonated mono- or oligosaccharide, amines, amino acids, or salts thereof.
  • the steps a, b and c are repeated.
  • the nucleophile is a nucleoside or nucleoside analog.
  • oligo- or polynucleosides can be prepared in an advantageous manner.
  • the steps a to c can be repeated until an oligo- or polynucleoside having the desired length or number of monomers, respectively, is received.
  • the method of the invention preferably comprises the further step(s) of d) deprotecting compound III and/or cleaving the residue R 1 from the linker or the solid phase. These steps are preferably performed under suitable conditions in order to safely release a compound I produced by the inventive method from the solid phase.
  • the method of the invention is preferably carried out under an inert gas atmosphere, preferably under nitrogen or argon gas.
  • the solid phase may be any solid phases, i.e. compounds being essentially insoluble under the conditions chosen.
  • Preferred solid phases are non-swellable or low-swellable materials, e.g. controlled pore glass (CPG) and macroporous polystyrene (MPPS).
  • CPG controlled pore glass
  • MPPS macroporous polystyrene
  • a preferred solid phase is a solid phase having a plurality of free amino groups.
  • Nucleosides or nucleoside analogs, nucleoside mono-, -di- and -triphosphates or mono-, di- and triphosphates of nucleoside analogs, and oligonucleosides may, for example, also be used as a nucleophile.
  • Example 1 General synthesis scheme for 5 '-modified oligonucleosides
  • oligonucleosides A general scheme for the synthesis of 5 '-modified oligonucleosides is depicted above.
  • B, X, Y, Z, (L), SP and R 6 are defined as above.
  • R 7 is a protecting group, e.g. 2-cyanoethyl.
  • Nu may be any nucleoside or nucleotide or phosphate or pyrophosphate, n is an integer > 0, e.g. 25.
  • a nucleoside or di-, tri- or oli- gonucleoside, as the case may be
  • is bound preferably via a linker L, e.g.
  • a cycloSai compound according to formula II is reacted with the unprotected oxygen at the 5' C atom of the sugar component of the immobilized nucleotide and the resulting compound is oxidized or sulfurized resulting in the corresponding phosphotriester, where Z may be O or Z.
  • a nucleoside is reacted as nucleophile Nu ⁇ with the immobilized cycloSai derivative, yielding a dinucleoside (or elongated oligonucleoside).
  • the reactions may be repeated until an oligonucleoside of the desired length is received.
  • the immobilized oligonucleoside may be deprotected and released from the solid phase SP.
  • Tetrasodium pyrophosphate decahydrate (2.62 g, 5.87 mmol) was dissolved in 60 mL Milli-Q water and eluted through a column filled with 200 g wet DOWEX 50WX8 (50-100 mesh), H + form.
  • the corresponding diphosphoric acid was collected in a flask and 7.60 g (11.72 mmol) tetra-n-butylammonium hydroxide, 40% w/w aqueous solution, added dropwise while strirring in an ice bath.
  • the subsequent triphosphorylation reaction was done by pushing 0.45 M bis(tetrabutyl- ammonium)dihydrogen pyrophosphate in DMF (dimethylformamide) through the synthesis column and let it left to react for 180 seconds. After this step the solution was pushed four more times through the column, 30 minutes between pushes. The total phosphorylation time was 2 hours.
  • the supported triphosphorylated oligonucleotide was washed with dry DMF, MeCN and dried under argon flow. 1.2.4 Deprotection and Purification
  • the corresponding phosphite was oxidized with 0.1 M oxidizer solution (I 2 , H 2 0, pyridine in THF), washed with dry DMF and MeCN and dried under argon flow.
  • the polystyrene bound nucleotide was transferred to an eppendorf vial and phos- phorylated with 1 mL of the previously described 0.45 M solution of

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Abstract

La présente invention concerne un procédé pour produire des conjugués de nucléoside à pont phosphate, en particulier des poly- ou oligonucléosides. Dans le procédé, un dérivé de cyclosaligényle immobilisé d'un nucléoside, un nucléotide, un poly- ou oligonucléotide, un poly- ou oligonucléoside, ou un analogue de celui-ci, est synthétisé, et la réaction consécutive avec un nucléophile produit le conjugué de nucléoside à pont phosphate souhaité, qui peut ensuite être libéré de la phase solide.
EP13701961.8A 2013-01-22 2013-01-22 Procédé pour la synthèse à base de phase solide de conjugués de nucléoside à pont phosphate Withdrawn EP2948467A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2022218943A1 (fr) * 2021-04-12 2022-10-20 Stealth Labels Biotech Ab Phosphates nucléosidiques fluorescents

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DE102008044914A1 (de) 2008-08-08 2010-02-11 Universität Hamburg Verfahren zur Herstellung phosphatverbrückter Nucleosid-Konjugate
DE102009020261A1 (de) * 2009-05-07 2010-11-11 Universität Hamburg Verfahren zur festphasengestützten Herstellung phosphatverbrückter Nucleosid-Konjugate

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See references of WO2014114326A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022218943A1 (fr) * 2021-04-12 2022-10-20 Stealth Labels Biotech Ab Phosphates nucléosidiques fluorescents

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US20150361124A1 (en) 2015-12-17

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